PROJECT SUMMARY The transmission of macromolecules across biological membranes is a fundamental process in all cells. In the earliest studies of genetic exchange in bacteria dating back to the 1940's, the F plasmid (then termed `sex factor') was shown to self-transfer and, through recombination, mediate the transfer of the entire E. coli chromosome to recipient bacteria. In the ensuing ~75 years, studies established the broad medical importance of F and other mobile genetic elements (MGEs) in the shaping of bacterial genomes and as vectors for dissemination of antibiotic resistance and other fitness traits among bacterial populations. MGEs also encode conjugative pili or other cell surface adhesins, which promote intercellular contacts necessary for DNA transfer and establishment robust, antibiotic-resistant biofilm communities. MGEs are transmitted intercellularly through nanomachines termed type IV secretion systems (T4SSs). The T4SSs are present in most if not all bacterial species, where they have functionally diversified into two large subfamilies, the DNA transfer or conjugation systems and the `effector translocators' that translocate effector proteins into eukaryotic host cells as a critical feature of infection processes. Over the past 27 years, my group has used molecular, genetic and biochemical approaches to identify many mechanistic and architectural features of T4SSs, including the first view of the translocation route for a DNA substrate through a T4SS. We have consistently implemented emerging technologies, and just within the past 1 years we began to solve T4SS structures at unprecedented resolution by in situ cryoelectron tomography (CryoET). These new structures are significantly advancing the field, but also are raising important new questions relating to underlying mechanisms and signals governing i) assembly of envelope-spanning T4SS channels and conjugative pili, ii) early-stage substrate recruitment and processing reactions, and iii) establishment of direct contacts (mating junctions) with bacterial and eukaryotic cells. Moving forward, we will address these fundamental questions by (1) continuing to solve novel structures encoded by the E. coli F T4SS using in situ CryoET, biochemical fractionation, super-resolution fluorescence microscopy, and single-particle CryoEM, (2) defining contributions of the newly visualized ATPase energy center positioned at the channel entrance in binding and unfolding substrates and dissociating accessory factors using in vivo and in vitro biochemistry and ultrastructural approaches, (3) exploring the roles of conjugative pili and cell surface adhesins in formation and disassembly of mating junctions using cytological, biochemical and biophysical approaches, and (4) exploiting our development of distinct model systems to identify mechanistic themes and specialized mechanisms. We will continue to draw on the expertise of our close collaborations for a `team-science' and multidisciplinary focus. Our studies will generate important new insights into the architecture, biogenesis, and mechanism of action of the T4SS superfamily. These findings will lead to major paradigm shifts in this field, and set the stage for design of intervention therapies.